In several tissues a coupling between glycolysis and Na+,K+-ATPase has been observed. The purpose of these studies was to investigate the coupling of glycolysis and Na+,K+-ATPase in Rous transformed Hamster cells, Ehrlich ascites tumor cells and A6 cultured cells. The rate of Na+,K+ATPase was estimated by the initial rate of ouabain-sensitive K+influx after K+ reintroduction to K+-depleted cells. Experiments were performed on HTcBH and Ehrlich cells producing ATP via oxidative phosphorylation alone (i.e. lactate sole substrate), glycolysis alone (i.e. glucose as substrate in the absence of oxygen or with antimycin A), or glycolysis and oxidative phosphorylation (i.e. glucose as substrate in the presence of oxygen). The cells produced ATP at approximately the same rate under all of these conditions, but the initial rate of K+-influx was approximately two-fold higher when ATP was produced from glycolysis. Changes in cell Na+ due to other transport processes related to glycolysis, such as Na+-H+ exchange, Na+-glucose cotransport and K+-H+ exchange were ruled out as mediators of this effect on Na+,K+-ATPase. These data suggest that glycolysis is more effective than oxidative phosphorylation in providing ATP to Na+,K+-ATPase in these cultured cells.